The present invention relates to screening methods, peptides, mimetics, and methods of use based on the surprising discovery and characterisation of an interaction between known proteins and the establishment that such interaction plays a key role in DNA repair, and thus numerous cellular processes of interest in therapeutic contexts. Two proteins in question are XRCC4 and DNA ligase IV. Interaction between XRCC4 and DNA-PKcs/Ku is also indicated.
The invention has arisen on the basis of the work of the present inventors establishing for the first time crucial information about XRCC4. Some information was available on the physiological function of this protein, it having been implicated in the Ku-associated DNA double-strand break repair (KADR) apparatus. However, very little was known about its biological activity and what its role in the KADR apparatus actually is. Prior to the making of the present invention it was not feasible to provide assays useful as primary screens for inhibitors of XRCC4.
Furthermore, the inventors"" new cloning work has identified a yeast homologue of mammalian DNA ligase IV. No physiological function has previously been assigned to mammalian DNA ligase IV, but the inventors"" yeast work, including analysis of the effect of knock-out mutation in yeast, now establishes the physiological relevance of DNA ligase IV and thus provides indication of therapeutic contexts in which modulation of its function can be effected.
The work disclosed herein establishing interaction between XRCC4 and DNA ligase IV, interaction between XRCC4 and DNA-PKcs/Ku, and also a biological role for such interactions, now gives rise to screening methods for identifying compounds which affect the interaction, particularly those which interfere with it, and which may affect or modulate particular aspects of cellular DNA repair activity, useful in a therapeutic context, for example in the treatment of proliferative disorders, cancers and tumours, disorders involving retroviruses such as AIDS, human adult T-cell leukemia/lymphoma, Type I diabetes and multiple sclerosis, and also in radiotherapy. Furthermore it gives rise to the rational design of peptides or mimetics or functional analogues which fulfil this function.
One of the most dangerous forms of damage that can befall a cell is the DNA double-strand break (DSB), which is the principal lethal lesion induced by ionizing radiation and by radiomimetic agents. Consequently, cells have evolved highly effective and complex systems for recognizing this type of DNA damage and ensuring that it is repaired efficiently and accurately. Two major pathways have evolved to repair DNA DSBs in eukaryotes, homologous recombination and DNA non-homologous end-joining (NHEJ).
Much of what is currently known about DNA NHEJ in mammalian systems has been obtained through studies of a series of mutant rodent cell lines that were identified originally as being hypersensitive towards ionizing radiation and which display severe defects in DNA DSB repair (reviewed in Jeggo et al., 1995; Roth et al., 1995). Characterisation of these cell lines has revealed that they fall into three complementation groups, termed. IR4, IR5 and IR7. The hamster cell line XR-1 defines IR4, IR5 consists of a number of independently isolated hamster cell mutants, and IR7 contains the hamster cell line V3 and cells derived from the severe combined immune-deficient (scid) mouse. Various studies have shown that IR4, IR5 and IR5 cells are defective in antibody and T-cell receptor V(D)J recombination.
Considerable effort has been directed towards establishing the nature of the gene-products defective in cells of IR4, IR5 and IR7, and determining how they function in DNA NHEJ. As a result of such studies, it was shown that cells of IR5 and IR7 are deficient in components of the DNA-dependent protein kinase (DNA-PK) (Ku80 and DNA-PKcs, respectively). DNA-PK is a nuclear protein Ser/Thr kinase that displays the unusual property of being activated upon binding to DNA DSBs or other perturbations of the DNA double-helix (Jackson, 1997). In light of the biochemical properties of DNA-PK which have been established, an attractive hypothesis is that this enzyme serves as a DNA damage sensor in vivo.
In contrast to cells of IR5 and IR7, XR-1 cells of IR4 are not deficient in a DNA-PK component, as evidenced by the fact that extracts of these cells have normal DNA end-binding activity (Getts and Stamato, 1994; Rathmell and Chu, 1994; Finnie et al., 1996) and DNA-PK activity (Blunt et al., 1995), and that expression of neither Ku80 nor DNA-PKcs complements the V(D)J recombination or radiosensitivity defects of XR-1 cells (Taccioli et al., 1994; Blunt et al., 1995). Instead, it has been shown that DNA from human chromosome region 5q13-14 complements XR-1 cells, the complementing gene being termed XRCC4 (Otevrel and Stamato, 1995).
Furthermore, (Li et al., 1995) have identified the XRCC4 gene recently through its ability to confer normal V(D)J recombination activity and partially restore the DSB repair defect on XR-1 cells, and have demonstrated that the XRCC4 locus is deleted in XR-1 cells.
Interestingly, XRCC4 encodes a small 334 amino acid residue protein of calculated molecular weight of 38 kDa, and the human and mouse homologues of this protein have been shown to be approximately 75% identical (Li et al., 1995). Perhaps surprisingly, however, sequence analyses reveal that XRCC4 is not significantly related to any previously-characterized proteins. Therefore, although it is clear that XRCC4 plays a crucial role in DNA DSB repair and V(D)J recombination, the cloning and sequencing of the cDNA for this factor has so far provided little clue to its mechanism of action.
The Li et al. paper is the only paper published on the XRCC4 protein as such prior to the priority date of the present invention. It reports that XRCC4 is not related to any other proteins and so its sequence gives no clear clues as to its function. Prior to the present work, therefore, the only assays available for XRCC4 were cellular radiosensitivity and cellular V(D)J recombinationxe2x80x94assays that cannot be used as primary screens for inhibitors. Consequently, it was impossible to conceive of any biochemical screen for the activity of this factor.
It should be noted too that the Li et al. paper does not provide any evidence that XRCC4 is a nuclear protein (shown herein) and discusses on page 1084 that XRCC4 has putative sites for cytoplasmic protein tyrosine kinases. Thus, it is clear that there really was nothing known about how this protein might act.
The present inventors have shown that XRCC4 exists, at least in part, in the cell nucleus and demonstrated convincingly that it interacts with DNA ligase IV, and also DNA-PKcs/Ku. Evidence is provided herein in the experimental section, with confirmation being provided also by Mizuta et al., 1997. Grawunder et al, 1997 has also provided evidence of interaction between XRCC4 and DNA ligase IV. See also the inventors"" publications Teo and Jackson, 1997 and Critchlow et al. 1997.
DNA ligases are catalysts which join together Okazaki fragments during lagging strand DNA synthesis, complete exchange events between homologous duplex DNA molecules, and seal single- or double-strand breaks in the DNA that are produced either by the direct action of a DNA damaging agents or by DNA repair enzymes removing DNA lesions (for review, see Lindahl and Barnes, 1992). In contrast to prokaryotic and yeast systems, where only a single species of DNA ligase has been previously been described (Johnston and Nasmyth, 1978), four biochemically distinct DNA ligases have been identified in mammalian cells (Tomkinson et al., 1991; Wei et al., 1995; Robins and Lindahl, 1996). In vitro assays, and studies of yeast and human cells containing mutated alleles of DNA ligase I suggest that this enzyme joins Okazaki fragments during DNA replication (Henderson et al., 1985; Malkas et al., 1990; Tomkinson et al., 1991; Barnes et al., 1992; Li et al., 1994; Prigent et al., 1994; Waga et al., 1994). Furthermore, the sensitivity of DNA ligase I mutant cells to ultraviolet (UV) irradiation and some DNA damaging agents suggests that DNA ligase I is involved in nucleotide excision repair and base excision repair (Henderson et al., 1985; Lehmann et al., 1988; Malkas et al., 1990; Tomkinson et al., 1991; Barnes et al., 1992; Li et al., 1994; Prigent et al., 1994; Waga et al., 1994).
Much less, however, is known about the function of the other three mammalian DNA ligases. It is currently unclear whether DNA ligase II and III arise from separate genes or by alternative splicing of the same gene (Roberts et al., 1994; Wang et al., 1994; Husain et al., 1995). However, ligase II is induced in response to alkylation damage (Creissen and Shall, 1982), suggesting a role in DNA repair. Similarly, the elevation of a splice variant of ligase III (ligase III-xcex2) levels in spermatocytes undergoing meiotic recombination (Chen et al., 1995; Husain et al., 1995; Mackey et al., 1997) and the association of another splice variant (ligase III-xcex1) with the DNA repair protein XRCC1 (Caldecott et al., 1994; Thompson et al., 1990) are consistent with this enzyme joining DNA strand breaks to complete DNA recombination and repair (Jessberger et al., 1993). Indeed, DNA ligase III, when present in a complex with XRCC-1, can reconstitute the ligation event necessary to complete base excision repair in vitro (Kubota et al., 1996).
A fourth enzyme, DNA ligase IV, has been purified recently from human cells and has distinct biochemical properties from other ligases (Robins and Lindahl, 1996). The physiological function of mammalian ligase IV is, however, unknown.
In most prokaryotes there is only one DNA ligase, and this enzyme catalyses all the DNA-joining events during replication, recombination and repair (Lindahl and Barnes, 1992). Similarly, genetic and biochemical data have suggested that there is only one DNA ligase in Saccharomyces cerevisiae (Lindahl and Barnes, 1992), although fractionation of yeast cell extracts has given an indication of a second DNA ligase activity (Tomkinson et al., 1992).
The present inventors searched for DNA ligase homologues in the S. cerevisiae genome, which was completely sequenced recently (Goffeau et al., 1996; Oliver, 1996). These searches identified a hitherto uncharacterized open reading frame (ORF) with sequence similarity along its entire length to mammalian DNA ligase IV. The experimental section below describes the effects of disrupting this gene, which the inventors have termed LIG4, on DNA replication, homologous recombination, and DNA repair in response to a variety of DNA-damaging agents. These studies show that LIG4 plays a crucial role in DNA double-strand break repair via the non-homologous end-joining (NHEJ) pathway but does not have an essential role in other DNA repair pathways studied.
Furthermore, it is shown that LIG4 functions in the same DNA repair pathway that utilizes the DNA end-binding protein Ku. However, the phenotype of lig4 mutant yeasts is not identical to those of yeasts disrupted for Ku function, revealing that Ku has additional roles in genome maintenance.
In summary, XRCC4 was known to be involved somehow in Ku-associated DNA double-strand break repair (KADR), but its biological activity was obscure. The present inventors have established for the first time biological activity of XRCC4, that is binding to DNA ligase IV. Furthermore, the physiological relevance of DNA ligase IV was not known. The inventors have now established that DNA ligase IV is important for double-strand DNA break repair via non-homologous end joining (NHEJ)xe2x80x94by unexpectedly identifying and cloning, then mutating, a yeast homologue gene and by establishing strong interaction between XRCC4 and DNA ligase IV.
The inventors have also established that XRCC4 interacts with DNA-PKcs/Ku, and shown that DNA-PKcs is able to phosphorylate XRCC4.
Based on this and other work described below, the present invention in various aspects provides for modulation of interaction between XRCC4 and DNA ligase IV.
Various aspect the present invention provide for the use of XRCC4 and DNA ligase IV in screening methods and assays for agents which modulate interaction between XRCC4 and DNA ligase IV.
Further aspects provide for modulation of interaction between XRCC4 and DNA-PKcs/Ku and use of these molecules in screening methods and assays for agents which modulate interaction between XRCC4 and DNA-PKcs/Ku. For simplicity, much of the present disclosure refers to XRCC4 and DNA ligase IV. However, unless the context requires otherwise, every such reference should be taken to be equally applicable to the interaction between XRCC4 and DNA-PKcs/Ku.
Methods of obtaining agents able to modulate interaction between XRCC4 and DNA ligase IV (or, it must be remembered, XRCC4 and DNA-PKcs/Ku) include methods wherein a suitable end-point is used to assess interaction in the presence and absence of a test substance. Detailed disclosure in this respect is included below. It is worth noting, however, that combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate bind to and/or activity of a polypeptide. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances.
Appropriate agents may be obtained, designed and used for any of a variety of purposes.
One is anti-tumour or anti-cancer therapy, particularly augmentation of radiotherapy or chemotherapy. Ionizing radiation and radiomimetic drugs are commonly used to treat cancer by inflicting DNA damage. Cells deficient in DNA repair, particularly the KADR pathway, are hypersensitive to ionizing radiation and radiomimetics. Evidence provided herein shows the KADR pathway involves XRCC4 and DNA ligase IV, indicating that inhibition of their function, e.g. by inhibiting their interaction, will have an effect on the KADR pathway, DNA repair and cellular sensitivity to ionizing radiation and radiomimetics.
Another is the potentiation of gene targeting and gene therapy. Inhibition of KADR may be used to increase efficiencies of gene targeting, of interest and ultimate use in gene therapy. Two ways exist for repairing DNA double-stranded breaks (DSBs). One is through the process of illegitimate recombination (also known as DNA non-homologous end-joining or NHEJ) and this is catalysed by the KADR system now known to involve XRCC4 and DNA ligase IV. The other system is the process of homologous recombination, whereby the damaged DNA molecule exchanges information with an undamaged homologous partner DNA molecule. In mammalian cells, the illegitimate pathway tends to predominate. Inhibiting the KADR system will make the proportion of DSBs repaired by homologous recombination increase. Thus, anti-KADR factor agents, including those provided in accordance with the present invention, will have this effect. Homologous gene targeting is used in making knock-out mice and other transgenic animals but it is not very efficient, so increasing this efficiency in accordance with the present invention will be highly beneficial. Ultimately, gene therapist wish to precisely replace the mutated gene with a functional one. At present just to get the functional gene to integrate anywhere in the genome is the priority, but the long-term aim is for integration at the right site. KADR (e.g. XRCC4 and/or ligase IV, or XRCC4 and/or DNA-PKcs/Ku) inhibitors therefore have a great therapeutic potential in such context.
A further, related, purpose is in anti-retroviral therapy, since DNA repair pathways such as involving KADR and the components XRCC4 and DNA ligase IV are involved in effecting retroviral and retrotransposon integration into the genome of a host cell. Retroviruses are of considerable risk to the health of humans and animals, causing, inter alia, AIDS, various cancers and human adult T-cell leukemia/lymphoma. Integration of retroviral DNA into the genome is essential for efficient viral propagation and may be targeted by inhibition of DNA repair pathway components.
Additionally, modulators of KADR components such as XRCC4 and DNA ligase IV, DNA-PKcs/Ku, may be used in modulation of immune system function, since such factors are required for generation of mature immunoglobulin and T-cell receptor genes by site-specific V(D)J recombination.
Compounds which stabilize the interaction between two components, such as XRCC4 and DNA ligase IV, or XRCC4 and DNA-PKcs/Ku, and which may up-regulate activity, may be screened for using assays in which conditions are too harsh for the relevant interaction. Agents which stabilize the interaction may be identified. One alternative is to screen for substances that enhance DNA ligase IV catalytic activity, which may be determined as discussed elsewhere. An up-regulator of activity may be used to potentiate DNA repair further, and this may be in normal individuals, with possible long-term beneficial effects bearing in mind that many of the common manifestations of ageing arise through the gradual and inexorable accumulation of mutations in somatic cells. Up-regulators may be used in treating patients who are debilited in the KADR pathway or other DNA repair pathway.
Interaction between XRCC4 and DNA ligase IV, or XRCC4 and DNA-Pkcs/Ku may be inhibited by inhibition of the production of the relevant protein. For instance, production of one or more of these components may be inhibited by using appropriate nucleic acid to influence expression by antisense regulation. The use of anti-sense genes or partial gene sequences to down-regulate gene expression is now well-established. Double-stranded DNA is placed under the control of a promoter in a xe2x80x9creverse orientationxe2x80x9d such that transcription of the xe2x80x9canti-sensexe2x80x9d strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the xe2x80x9csensexe2x80x9d strand of the target gene. The complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works.
Another possibility is that nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific sitexe2x80x94thus also useful in influencing gene expression. Background references for ribozymes include Kashani-Sabet and Scanlon, 1995, Cancer Gene Therapy, 2(3): 213-223, and Mercola and Cohen, 1995, Cancer Gene Therapy, 2(1), 47-59.
Thus, various methods and uses of modulators, particularly inhibitors, of XRCC4 and DNA ligase IV, or XRCC4 and DNA-PKcs/Ku, interaction and/or activity are provided as further aspects of the present invention. The purpose of disruption, interference with or modulation of interaction between XRCC4 and DNA ligase IV, and/or XRCC4 and DNA-PKcs/Ku, may be to modulate any activity mediated by virtue of such interaction, as discussed above and further below.